The mammalian cochlea has two types of hair cells. Cochlear hair cells are non-neuronal epithelial cells that transduce acoustic signals. Outer hair cells (OHCs) are responsible for the exquisite sensitivity and frequency-resolving capacity of the normal mammalian hearing organ, the ear; they provide local mechanical amplification (the xe2x80x9ccochlear amplifierxe2x80x9d) in the form of feedback (1987, Ashmore, et al., J. Physiol. (London), 388:323-347). In contrast, inner hair cells (IHCs) convey auditory information to the brain (Dallos, P., Overview: Cochlear Neurobiology, pages 1-43, Springer, N.Y., 1996). OHCs have cylindrical somata of constant diameter and variable length. It is generally believed that the mammalian cochlea owes its remarkable sensitivity, frequency selectivity, and various complex nonlinear properties to a mechanical feedback action by OHCs.
In response to membrane potential change, the OHC rapidly alters its length and stiffness (1985, Brownell et al., Science, 227:194-196). These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by IHCs. These somatic shape changes may be up to 5% of the cell length; The cell shortens when depolarized and lengthens when hyperpolarized. Length changes do not depend on either ATP or Ca2+(1988, Holley et al., Proc. R. Soc. Lond. Ser. B. Biol. Sci., 232:413-429.) and they can be elicited with unchanging amplitude at microsecond rates up to high audio frequencies. Motile responses are accompanied by charge movement, reflected in nonlinear capacitance, akin to the translocation of gating charges of voltage-gated ion channels (1991, Santos-Sacchi et al., J. Neurosci., 11:3096-3110). This nonlinear capacitance is widely used as a xe2x80x9csignaturexe2x80x9d of the electromotile process. Motility is also accompanied by axial stiffness change of the cell. By virtually any test, electromotility and electrically-induced stiffness changes can be correlated with each other and they are collectively described as voltage-dependent mechanical changes of the OHC, heretofore called electromechanics. These observations make it apparent that the fast mechanical changes in OHCs are powered by a novel molecular motor, fundamentally different from other biological force generators, such as the myosin, kinesin or dynein families. The OHC molecular motor performs direct, rapid, reversible electromechanical conversion.
Despite extensive studies of cellular and biophysical mechanisms of OHC function, very little is known about the genes and molecular events involved in OHC function. OHC electromotility is the likely result of the concerted action of a large number of independent molecular motors that are closely associated with the cell""s basolateral membrane, possibly by the densely packed 10 nanometer particles seen therein.
There have been some suggestions as to the identity of these motor molecules. Based on similarities between the cortical structure of erythrocytes and OHCs, it has been proposed that the motor molecule is a modified anion exchanger. Because their shallow voltage dependence matches that of charge movement in OHCs, transporters have been favored, as opposed to modified voltage-dependent channels, as likely candidates. A recent suggestion is that the motor is related to a fructose transporter, GLUTS. Until the present invention, molecular identification of the motor protein has not been achieved.
The invention includes an isolated polynucleotide comprising a portion that anneals with high stringency with at least twenty consecutive nucleotide residues of a coding region of a mammalian pres gene.
In one aspect, the mammalian pres gene comprises a nucleotide sequence listed in SEQ ID NO:2 or SEQ ID NO:4.
In another aspect, the coding region is one other than a coding region corresponding to any of exons 1-6 of the human pres gene.
The invention also includes an isolated polynucleotide comprising the coding regions of a mammalian prestin gene, wherein the coding regions of the prestin gene are at least 75% homologous with the coding region of at least one of the gerbil and murine prestin gene.
In a preferred aspect, the isolated polynucleotide comprises a promoter/regulatory region operably linked with the coding regions.
An isolated mammalian prestin protein is also encompassed by the invention. In one aspect, the mammalian prestin protein is isolated from a gerbil, a mouse, or a human. In another aspect, the protein has an amino acid sequence listed in SEQ ID NO:1 or SEQ ID NO:3. Preferably, the protein is substantially purified.
Also contemplated by the invention is an isolated antibody which binds specifically with a mammalian prestin protein.
The invention includes a method of alleviating a hearing disorder in a mammal afflicted with the disorder comprising providing a mammalian prestin protein to cochlear outer hair cells of the mammal, thereby alleviating the disorder. In one aspect, the protein is provided to the outer hair cells by providing a nucleic acid vector comprising a polynucleotide encoding the protein to the outer hair cells.
A method of rendering the surface area of a lipid bilayer susceptible to modulation by membrane potential is also envisaged in the invention. The method comprises providing a mammalian prestin protein to the bilayer, whereby the bilayer is rendered susceptible to modulation by membrane potential. In an aspect of the invention, the lipid bilayer is the plasma membrane of a cell.
In another aspect, the protein is provided to the lipid bilayer by providing an expressible nucleic acid vector comprising a polynucleotide encoding the protein to the cell and then expressing the protein in the cell.
The invention also includes a method of modulating the surface area of a lipid bilayer. The method comprises providing a mammalian prestin protein to the bilayer and modulating the membrane potential, thus modulating the surface area of the bilayer.
A method for modulating stiffness of a lipid bilayer surrounding a relatively fixed volume is also within the scope of the invention. The method comprises providing a mammalian prestin protein to the bilayer, modulating the membrane potential, and thus, modulating bilayer stiffness.
The invention also encompasses a method of modulating the volume of a porous bilammelar lipid vesicle. The method comprises providing a mammalian prestin protein to the bilayer and modulating the membrane potential, thereby modulating the volume of the vesicle.
The invention further includes a method for generating a force between two surfaces. The method comprises interposing a structure having a lipid membrane comprising prestin and enclosing a relatively fixed volume of fluid between the surfaces, restraining the ability of the structure to expand in a direction at least partially parallel to at least one of the surfaces, and altering the membrane potential of the lipid membrane. The structure impacts upon the two surfaces and a force is generated between them.
The invention also describes a method for generating an electrical impulse by applying a mechanical force to the prestin protein. The method comprises applying a mechanical stress on the prestin protein, such that the protein creates an electrical impulse in response to the mechanical force.